CN116745426A - ADAMTS13 variants with increased escape rate or activity against autoantibodies - Google Patents

ADAMTS13 variants with increased escape rate or activity against autoantibodies Download PDF

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Publication number
CN116745426A
CN116745426A CN202180077809.2A CN202180077809A CN116745426A CN 116745426 A CN116745426 A CN 116745426A CN 202180077809 A CN202180077809 A CN 202180077809A CN 116745426 A CN116745426 A CN 116745426A
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residue
replaced
adamts13
leu
gly
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南铉子
黄成虎
郭曦天
崔嘉熙
金洙镕
金侑映
李镕愍
李砦牧
慎善惠
权瑛银
曹胜贤
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GREEN CROSS CORP
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GREEN CROSS CORP
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Abstract

The present invention relates to ADAMTS13 mutant proteins having increased escape rates for autoantibodies, and compositions for their use in the prevention or treatment of thrombotic diseases. By effectively avoiding representative autoantibodies known to have high binding affinity to the main domain of ADAMTS13, the ADAMTS13 variant proteins of the present invention can be used as effective therapeutic compositions for a variety of thrombotic diseases such as TTP (thrombotic thrombocytopenic purpura) and the like whose main causes are the presence of these autoantibodies, and can stably maintain their biological activity when administered into the body. In addition, since new sites recognized by autoantibodies are identified within ADAMTS13, the present invention can be effectively used to screen for new ADAMTS13 variants with increased escape rates of autoantibodies by applying a combination of multiple mutations within the corresponding sites.

Description

ADAMTS13 variants with increased escape rate or activity against autoantibodies
Technical Field
The present invention relates to variants of ADAMTS13 having reduced or high enzymatic activity against autoantibodies and compositions for their use in the treatment of thrombotic disorders.
Background
Thrombotic thrombocytopenic purpura (thrombotic thrombocytopenic purpura, TTP) is a rare blood disease that belongs to thrombotic microangiopathy, which forms blood clots in small blood vessels throughout the body, which can lead to death if not treated immediately. The incidence is known to be 1.5 to 6 out of every 100 thousands of people per year, and is high mainly in adults and women with an average age of 40 years (Miesbach et al, 2019). Its pathological features include thrombocytopenia, erythropenia, increased Hematocrit (HCT), etc., and due to blood clots, dysfunction is known to occur in many organs such as the kidney, heart, brain. Symptoms include bruising, fever, weakness, dyspnea, blurred consciousness, headache, etc. (Hovinga et al, 2017).
Thrombotic Thrombocytopenic Purpura (TTP) is divided into two classes: congenital TTP (cTTP) which causes congenital ADAMTS13 functional defects due to dysfunction of genes encoding ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 biotif, membrane 13) (disintegrin and metalloprotease with thrombospondin type 1motif, member 13); and acquired TTP (atttp), which is caused by a decrease in ADAMTS13 activity due to acquired. In general, TTP is diagnosed when ADAMTS13 activity is less than 10% of normal TTP. The aTTP is reported to be caused by bacterial infection, certain drugs, autoimmune diseases (e.g., lupus), pregnancy, etc., and as one of the main mechanisms of pathogenesis, inhibition of ADAMTS13 activity by autoantibodies recognizing ADAMTS13 has been reported (GARD program, 2018). ADAMTS13 enzymes function in the following ways: large multimers of von willebrand factor (von Willebrand factor, vWF) are broken down into smaller units, where antibodies found in aTTP patients bind to ADAMTS13 and inhibit its function, and prevent vWF degradation, and ultimately lead to blood clots and platelet overproduction, leading to disease.
As standard treatment for cTTP patients, complementary treatment to provide deficient lytic enzymes by injection of fresh frozen plasma (fresh frozen plasma) was used, whereas for atttp patients, neutralizing antibodies were removed by Plasma Exchange (PEX) for symptomatic relief. In the case of aTTP patients, combined administration of immunosuppressants (prednisolone), corticosteroids (coticosteroid), etc.) to improve therapeutic effect, prevent relapse, and rituximab (which induces B cell death) can be used to reduce the production of neutralizing antibodies. Although the number of plasma exchanges depends on the severity of the disease and the progress of the symptoms, several plasma exchanges are usually required to normalize the platelet count. Current treatments have limitations such as the inconvenience of repeated plasma exchanges and the increased susceptibility to infection resulting from the use of immunosuppressants.
Thus, to overcome the limitations of conventional therapies against TTP, the present inventors prepared recombinant ADAMTS13 protein variants that remained active by effectively escaping binding to patient autoantibodies even in the presence of autoantibodies, and confirmed that recombinant ADAMTS13 protein variants can be effectively used as effective therapies against TTP by efficacy evaluation in a TTP mouse model.
Throughout this specification, numerous journal articles and patent documents are referenced and their references are pointed out. The disclosures of the cited journal articles and patent documents are incorporated herein by reference in their entireties to more clearly describe the level of skill in the art to which the present invention pertains and the details of the present invention.
Disclosure of the invention
Technical problem
The present inventors have made extensive efforts to develop effective and basic methods for treating a variety of ADAMTS13 dysfunctional diseases, which are mostly among refractory rare diseases. As a result, the present inventors have found that when the core region of ADAMTS13 recognized by autoantibodies is identified and some amino acids in these regions are substituted, binding to the autoantibodies is blocked, and activity against vWF degradation and thrombosis inhibition is maintained, and thus, it is useful as an effective therapeutic composition for various diseases caused by excessive thrombosis, including Thrombotic Thrombocytopenic Purpura (TTP), leading to completion of the present invention.
Accordingly, it is an object of the present invention to provide ADAMTS13 variant proteins or functional fragments thereof.
It is another object of the present invention to provide a composition for preventing or treating thrombotic diseases.
It is another object of the present invention to provide methods for screening for ADAMTS13 variant proteins with increased escape rates against ADAMTS13 autoantibodies.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the accompanying drawings.
Solution to the problem
According to one aspect of the present invention, there is provided an ADAMTS13 (thrombospondin type 1 motif-containing desmin and metalloprotease, member 13) variant protein or functional fragment thereof comprising a substitution of one or more amino acid residues selected from the group consisting of: residue 85, residue 93, residue 126, residue 135, residue 278, residue 282, residue 308, residue 314, residue 317, residue 334, residue 364, residue 376, residue 413, residue 427, residue 452, residue 465, residue 567, residue 578, residue 585, residue 589, residue 607, residue 608, residue 609, residue 612, residue 618, residue 630, residue 635, residue 643, residue 651, residue 655, residue 656, residue 655, and residue 655 of SEQ ID NO.
The present inventors have made extensive efforts to develop effective and basic methods for treating a variety of ADAMTS13 dysfunctional diseases, which are mostly among refractory rare diseases. As a result, the present inventors have found that when the core region of ADAMTS13 recognized by autoantibodies is identified and some amino acids in these regions are substituted, binding to the autoantibodies is blocked, and vWF degradation and thrombotic inhibition activity is maintained, and thus, they are useful as effective therapeutic compositions for various diseases caused by excessive thrombosis, including Thrombotic Thrombocytopenic Purpura (TTP).
The term "protein" as used herein refers to a linear molecule formed by binding amino acid residues to each other through peptide bonds.
The term "functional fragment" as used herein refers to an analog of a full-length protein, which is a fragment of the full-length protein in which some amino acid residues have been deleted, preserving the original biological activity and function of the full-length protein.
According to the invention, SEQ ID NO. 1 is an amino acid sequence of an ADAMTS13 protein consisting of 1427 amino acids. Thus, an ADAMTS13 variant protein or functional fragment thereof of the invention can be a full-length (1427a.a.) ADAMTS13 protein or an ADAMTS13 variant in which the above-listed variants are introduced into its functional fragment (comprising the 75 th to 685 th region). Some fragments comprising the 75 th to 685 th regions may be, for example, 1 to 685 (685 a.a.) or 75 to 685 (611 a.a.).
SEQ ID NO. 1 is an amino acid sequence substantially comprising the ADAMTS13 proteins and functional fragments thereof of the present invention, which also comprises an amino acid sequence exhibiting substantial identity to the above sequences. Significant identity (substantial identity) refers to an amino acid sequence that exhibits at least 70% homology, particularly at least 80% homology, more particularly at least 90% homology, and most particularly at least 95% homology to the amino acid sequence described above when analyzed after aligning the amino acid sequence to the amino acid sequence described above as much as possible using algorithms commonly used in the art.
The term "autoantibody" as used herein refers to an antibody that is produced by the individual's own immune system, recognizes and targets its own protein, one of the immunoglobulins comprising one or more variable domains that bind to an epitope of an antigen so as to be able to specifically recognize that antigen. The presence of autoantibodies results in a decrease or loss of endogenous function or biological activity of the proteins specifically recognized by the corresponding autoantibodies, and thus becomes a cause of various diseases.
According to a specific embodiment of the invention, the ADAMTS13 variant protein is selected from each variant protein comprising amino acid residue substitutions at the following positions:
-residues 85 and 317; residue 612; two or more of residue 282, residue 465 and residue 672; residue 635; residues 452 and 612; two or more of residues 278, 334 and 427; residue 618; residue 135; two or more of residues 126, 567 and 651; residue 413; residue 334; residue 314; two or more of residues 93, 364 and 376; residue 308; residue 656; residue 607; residues 612 and 624; residue 589; residues 650 and 656; residues 643; residue 585 and residue 658; two or more of residues 630, 654 and 664; four or more of residue 589, residue 608, residue 609, residue 624 and residue 655; residue 578; residue 585; residues 314 and 635; and residues 314 and 612.
According to one embodiment of the invention, in the variant positions used in the invention, the 85 th residue is replaced with Phe, the 93 rd residue is replaced with Val, the 126 th residue is replaced with Met, the 135 th residue is replaced with Ile, the 278 th residue is replaced with Ile, the 282 th residue is replaced with Ala, the 308 th residue is replaced with Lys, the 314 th residue is replaced with Thr, the 317 th residue is replaced with His, the 334 th residue is replaced with Thr or Val, the 364 th residue is replaced with Arg, the 376 th residue is replaced with Asp, the 413 th residue is replaced with Asp, the 427 th residue is replaced with Asn, the 452 th residue is replaced with Ile, the 465 th residue is replaced with Asp, the 567 th residue is replaced with Ser, the 585 th residue is replaced with Asn or Met, the 589 th residue is replaced with gin, the 607 th residue is replaced with Arg, the 608 th residue is replaced with Met, the 609 th residue is replaced with Leu, the 612 th residue is replaced with Phe or Tyr, the 618 th residue is replaced with Ser, the 624 th residue is replaced with Asp or Cys, the 630 th residue is replaced with Leu, the 635 th residue is replaced with Val, the 643 th residue is replaced with Phe, the 650 th residue is replaced with His, the 651 th residue is replaced with Asp, the 654 th residue is replaced with Gly, the 655 th residue is replaced with Val, the 656 th residue is replaced with Arg or His, the 658 th residue is replaced with His, the 664 th residue is replaced with Asn, and the 672 th residue is replaced with Val.
According to another aspect of the present invention there is provided a fusion protein comprising: (a) The ADAMTS13 variant proteins or functional fragments thereof of the present invention; and (b) an Fc region of an IgG4 immunoglobulin conjugated to (a) above.
The inventors have found that when an Fc region derived from an IgG4 immunoglobulin is conjugated to an ADAMTS13 variant protein found in the present invention, the in vivo stability is significantly improved while the activity of endogenous vWF cleavage and the activity of escaping neutralizing antibodies can be maintained, and in particular, the structural instability exhibited by an open form (open form) fragment in which a portion of the distal end has been removed is significantly improved.
According to a specific embodiment of the invention, the Fc region comprises a substitution of one or more amino acid residues selected from the group consisting of residue 22, residue 24 and residue 26 of SEQ ID NO. 2. More specifically, residue 22 is replaced with Tyr, residue 24 is replaced with Thr, and residue 26 is replaced with Glu, respectively.
According to the invention, SEQ ID NO. 2 is an Fc region derived from an IgG4 immunoglobulin (217a.a.). The present inventors have found that when the above-described ADAMTS13 variant protein or functional fragment thereof is fused to an Fc region [ IgG4 (YTE) ] derived from IgG4 immunoglobulin (in which residues 22, 24, and 26 are replaced with Tyr, thr, and Glu, respectively), the blood half-life of the ADAMTS13 variant protein or functional fragment thereof is maximized, and thus the physiological activity after administration can be maintained for a long period of time.
According to a specific embodiment of the invention, the fusion protein of the invention further comprises a hinge region of an IgG1 immunoglobulin between (a) and (b) above.
According to the invention, the hinge region derived from an IgG1 immunoglobulin may be represented by SEQ ID NO. 3 (15 a.a.).
According to another aspect of the present invention, the present invention provides the above-mentioned ADAMTS13 variant protein of the present invention or functional fragment thereof; or a nucleotide encoding the above fusion protein.
The term "nucleotide" as used herein has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides as basic building blocks in nucleic acid molecules include not only natural nucleotides but also analogues thereof in which sugar or base regions are modified. It will be apparent to those skilled in the art that the nucleotide sequences to be expressed for therapeutic, pharmaceutical production, etc. purposes are not limited in the present invention to the nucleotide sequences set forth in the appended sequence listing. Certain variations in nucleotides do not cause alterations in the protein. Such nucleic acids encompass all molecules having the following: functionally equivalent codons, codons encoding the same amino acid due to codon degeneracy, and codons encoding biologically equivalent amino acids.
In view of the above variants having biologically equivalent activity, the nucleotides to be used in the present invention are to be construed as including sequences exhibiting significant identity to the sequences depicted in the sequence listing. Significant identity refers to an amino acid sequence that exhibits at least 70% homology, at least 80% homology, more particularly at least 90% homology, and most particularly at least 95% homology with the amino acid sequence described above when analyzed after aligning the amino acid sequence with the amino acid sequence of the present invention as much as possible using algorithms commonly used in the art. Alignment methods for sequence comparison are disclosed in the art. Various methods and algorithms for alignment are described in Huang et al, comp.Appl. BioSci.8:155-65 (1992) and Pearson et al, meth.mol.biol.24:307-31 (1994). NCBI basic local alignment search tools (Basic Local Alignment Search Tool, BLAST) (Altschul et al, J.mol.biol.215:403-10 (1990)) are available from the national center for Biotechnology information (National Center for Biological Information, NCBI), and the like, and can be used in conjunction with sequencing programs such as blastp, blastn, blastx, tblastn and tblastx.
The nucleotides of the invention can be used in gene therapy to deliver the variant ADAMTS13 of the invention described above to a subject in gene form, or can be used to produce recombinant proteins in pharmaceutical form by expressing an ADAMTS13 variant protein in a host cell.
The term "expression" as used herein means that in order to allow a subject to express an exogenous (exogenous) gene or to introduce an exogenous gene using a gene vector to increase the natural expression level of an endogenous (endogenous) gene, the gene can be replicated within the cells of the subject as an extrachromosomal element or by completing chromosomal integration. Thus, the term "expression" is synonymous with "transformation", "transfection" and "transduction".
The term "gene vector" as used herein refers to any means of transporting a gene into a cell, and gene delivery has the same meaning as gene transduction in a cell. At the tissue level, the term gene delivery has the same meaning as gene propagation (spread). Thus, the gene delivery system of the present invention can be described as a gene penetration system and a gene delivery system.
The gene delivery system of the invention may be included in the form of an expression cassette, which is a polynucleotide construct comprising all the elements necessary for self-expression of the gene to be introduced. The expression cassette typically comprises a promoter operably linked to the gene, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication.
The gene delivery system used in the present invention may be applied to any gene delivery system used for conventional gene insertion, and may include, for example, plasmids, adenoviruses, adeno-associated viruses (AAV), retroviruses and lentiviruses, herpes simplex viruses, vaccinia viruses, liposomes and vesicles, but is not limited thereto.
According to another aspect of the present invention, there is provided a composition for preventing or treating thrombotic diseases, comprising as active ingredients: the ADAMTS13 variant proteins or functional fragments thereof of the present invention; the fusion protein; or a nucleotide encoding the same.
According to another aspect of the present invention there is provided a method for preventing or treating a thrombotic disorder comprising administering to a subject a composition comprising: the ADAMTS13 variant proteins or functional fragments thereof of the present invention; the fusion protein; or a nucleotide encoding the same, as an active ingredient.
The term "thrombotic disorder" as used herein refers to a systemic disorder in which blood flow is reduced or blocked due to thrombus generated by platelet aggregation in the microcirculation system of blood vessels, thereby causing ischemic injury to each organ such as kidney, heart and brain.
Excessive platelet aggregation and thrombus overproduction can occur when ADAMTS13 enzymatic activity is inhibited by neutralizing antibodies, and thus von willebrand factor (vWF) is not properly degraded. Accordingly, the ADAMTS13 variant proteins of the invention are useful as compositions for effectively preventing or treating a variety of thrombotic diseases, while maintaining or enhancing vWF degrading activity while efficiently escaping neutralizing antibodies.
The term "preventing" as used herein refers to inhibiting the occurrence of a disorder or disease in a subject who has never been diagnosed with the disorder or disease but who is likely to have the disorder or disease.
The term "treatment" as used herein refers to (a) inhibiting the progression of a disorder, disease or symptom; (b) alleviating a disorder, disease or symptom; or (c) elimination of a disorder, disease or symptom. When the composition of the present invention is administered to a subject, the composition plays a role in specifically recognizing and degrading vWF to block the generation of excessive thrombus, regardless of the presence or absence of neutralizing antibodies, thereby inhibiting, eliminating or reducing the progress of thrombotic diseases. Thus, the compositions of the present invention may be as such compositions for the treatment of these diseases or may be administered with other pharmacological ingredients and as therapeutic aids for the above-mentioned diseases. Thus, the terms "treatment" or "therapeutic agent" as used herein include the meaning of "supplemental treatment" or "therapeutic adjuvant
The term "administering" or variations thereof as used herein refers to directly administering to a subject a therapeutically effective amount of a composition of the invention such that the same amount is formed in the body of the subject.
The term "therapeutically effective amount" as used herein refers to the content of the composition, wherein the pharmacological component contained in the composition is sufficient to provide a therapeutic or prophylactic effect to the individual to whom the pharmaceutical composition of the invention is to be administered, and the term includes the meaning of "prophylactically effective amount".
The term "subject" as used herein includes, but is not limited to, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons, and rhesus monkeys. In particular, the subject of the invention is a human.
According to a particular embodiment of the invention, the thrombotic disorder that can be prevented or treated by the composition of the invention is thrombotic microangiopathy (thrombotic microangiopathy, TMA). More particularly, thrombotic Microangiopathy (TMA) is Thrombotic Thrombocytopenic Purpura (TTP), hemolytic uremic syndrome (hemolytic uremic syndrome, HUS), HELLP (Hemolysis, elevated Liver enzymes, and Low Platelet count) (hemolytic, liver enzyme elevated and thrombocytopenic) syndrome, preeclampsia (preeclampsia) and sickle cell disease (sickle cell disease), more particularly Thrombotic Thrombocytopenic Purpura (TTP) or sickle cell disease, even more particularly Thrombotic Thrombocytopenic Purpura (TTP).
When the composition of the present invention is prepared as a pharmaceutical composition, pharmaceutically acceptable carriers contained therein are those commonly used in formulations, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginic acid, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like, but are not limited thereto. In addition to the above ingredients, the pharmaceutical compositions of the present invention may also contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington' sPharmaceutical Sciences (19 th edition, 1995).
The pharmaceutical composition of the present invention may be administered orally or parenterally, in particular, it may be administered parenterally, and more particularly, it may be administered subcutaneously, transdermally or intravenously.
The appropriate dosage of the pharmaceutical composition of the present invention may be variously prescribed according to various factors such as formulation method, administration method, age, body weight, sex, health condition, food, administration time, administration route, excretion rate and reaction sensitivity of patients. The preferred dosage of the pharmaceutical composition of the present invention is 0.001mg/kg to 100mg/kg for adults.
The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating according to methods readily performed by those of ordinary skill in the art to which the present invention pertains using pharmaceutically acceptable carriers and/or excipients, or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.
According to another aspect of the present invention, there is provided a method for screening for ADAMTS13 variants with increased autoantibody escape rate, comprising the steps of:
(a) Preparing an ADAMTS13 variant in which one or more amino acid residues selected from the group consisting of: residue 85, residue 93, residue 126, residue 135, residue 278, residue 282, residue 308, residue 314, residue 317, residue 334, residue 364, residue 376, residue 413, residue 427, residue 452, residue 465, residue 567, residue 578, residue 585, residue 589, residue 607, residue 608, residue 609, residue 612, residue 618, residue 630, residue 635, residue 643, residue 650, residue 651, residue 655, residue 656, residue 658, residue 664, and residue 672 of the first sequence of the sequence listing; and
(b) Contacting an autoantibody against ADAMTS13 with the ADAMTS13 variant prepared in step (a) above;
wherein when the autoantibody binds to the ADAMTS13 variant with less affinity than to wild-type ADAMTS13, the ADAMTS13 variant is determined to be an ADAMTS13 variant with increased escape rate to the autoantibody.
According to the present invention, the present inventors have analyzed whether libraries of about 800 more variants obtained using random mutagenesis (Random mutagenesis) can bind to ADAMTS13 neutralizing antibodies; as a result, the present inventors have found that the positions of the amino acid residues in SEQ ID NO. 1 play an important role in binding to neutralizing antibodies, and that affinity for neutralizing antibodies can be controlled by variation at these positions. Thus, a variety of variants can be derived by the types of variations introduced by these residue positions found by the present inventors and combinations of variations thereof, and ADAMTS13 variant enzymes whose activity is not reduced by the presence of autoantibodies can be selected by measuring the binding of these variants to autoantibodies.
The term "autoantibody escape rate" as used herein refers to a value expressed as a percentage of the ratio of ADAMTS13 variants that do not bind to an autoantibody as compared to wild-type ADAMTS13, and a high escape rate against an autoantibody means that affinity against an autoantibody is low and thus unique biological activity (e.g., vWF degrading activity) of ADAMTS13 is not inhibited by the autoantibody. Thus, the term "ADAMTS 13 variant with increased autoantibody escape rate" can also be expressed as "ADAMTS 13 variant with reduced affinity for autoantibodies" or "ADAMTS 13 variant with reduced binding ability to autoantibodies".
According to the present invention, the escape rate of candidate variants against an autoantibody can be evaluated by measuring the binding between the ADAMTS13 variant prepared in step (a) above and the autoantibody. Binding to an autoantibody can be measured by a variety of methods, and one of the methods is a method comprising: antibodies to the antigen (or cells expressing the antigen) are incubated, unbound antibodies are removed (e.g., washed), and bound antibodies are detected with labeled antibodies bound thereto. The binding between an antigen and an antibody is typically mediated through the Complementarity Determining Regions (CDRs) of the antibody and epitopes of the antigen, and unlike random and non-specific binding of proteins, the specific three-dimensional structure of the antigen and variable domains allows precise binding of these two structures. Thus, if the autoantibodies found by the present invention are appropriately combined with an appropriate combination of variations in key reaction sites, the binding of the autoantibodies can be effectively blocked.
As a result of the measurement, candidate variants having reduced affinity or binding capacity for the autoantibody as compared to the wild type can be determined as ADAMTS13 variants having increased escape rate for the autoantibody.
The term "reduced affinity" as used herein refers to a significant reduction in binding between an ADAMTS13 variant and an autoantibody to such an extent that the inherent enzymatic activity of ADAMTS13 is increased to a measurable level, which can also be described as a "reduced binding capacity". In particular, it refers to a state in which binding is reduced by 20% or more, more particularly by 40% or more, and more particularly by 60% or more compared to the wild type.
According to a particular embodiment of the invention, step (a) above is carried out by substitution of one or more amino acid residues selected from the group consisting of: residue 85, residue 93, residue 126, residue 135, residue 278, residue 282, residue 308, residue 314, residue 317, residue 334, residue 376, residue 413, residue 427, residue 465, residue 567, residue 578, residue 585, residue 607, residue 609, residue 612, residue 624, residue 630, residue 643, residue 650, residue 654, residue 655, residue 656, residue 658, and residue 672.
Advantageous effects of the invention
The features and advantages of the present invention may be summarized as follows:
(a) The present invention provides ADAMTS13 variant proteins having increased escape rates for autoantibodies, and compositions for their use in the prevention or treatment of thrombotic disorders.
(b) The ADAMTS13 variant proteins of the present invention, by way of effectively escaping representative autoantibodies known to have high binding affinity to the main domain of ADAMTS13, can be used as effective therapeutic compositions for a variety of thrombotic diseases, such as Thrombotic Thrombocytopenic Purpura (TTP) in which autoantibodies are the primary cause, and can stably maintain biological activity when administered into the body.
(c) In addition, in the present invention, since a new site recognized by an autoantibody is also identified within ADAMTS13, a combination of a plurality of variations can be applied within the corresponding site and thus can be effectively used to screen for new ADAMTS13 variants with increased escape rate against an autoantibody.
Drawings
FIG. 1 shows a graph illustrating the results of construction and validation of mutation rates for a library of human ADAMTS13 variants using random mutagenesis. FIG. 1a is a schematic representation of the library construction process by random mutagenesis. Random variation was induced in the MDTCS region or S domain by error-prone PCR (EP PCR), inserted into a recipient vector, cloned, and subsequently transformed into e.coli to ensure colony formation. DNA was extracted from colonies and the mutated nucleotide sequence was confirmed by Sanger sequencing analysis. FIG. 1b shows a graph illustrating the results of a sequencing analysis. The total variability and variant forms of the library constructed from the targeted MDTCS or S domains were confirmed, respectively.
Fig. 2 shows a graph illustrating the results of confirming the binding site of each domain of ADAMTS13 binding to the neutralizing antibody. FIG. 2a shows a schematic representation of the preparation of six ADAMTS13 fragments (or wild-type full-length ADAMTS 13) that combine multiple domains to confirm the expression rate of ADAMTS13 or the binding site of neutralizing antibodies. FIG. 2b shows the results of western blotting with anti-V5 antibodies after immunoprecipitation by using protein-mixed protein A-Sepharose expressed in each ADAMTS13 domain and each neutralizing antibody. The "input" shows the protein expression level of each domain. FIG. 2c is a schematic representation of the binding sites of ADAMTS13 neutralizing antibodies, illustrating the specific binding of Ab 4-16 antibodies to the S domain, ab 67 antibodies to the D domain, and Ab 66 antibodies to the T2-T8 domain, respectively.
Fig. 3 shows a graph illustrating the results of screening for variants that escape anti-ADAMTS 13 antibodies or variants that have activity superior to wild-type ADAMTS 13. Escape rates and ADAMTS13 activity of Ab 4-16 antibodies or Ab 67 antibodies were measured in wild-type clones and ADAMTS13 variants. The relative activity was calculated by confirming the specific titers of wild-type clones and variants and substituting them into the following equation: relative activity (%) =specific potency of variant/specific potency of wild-type ADAMTS13 x 100.
Fig. 4 shows the results of confirming the escape rate of 12 full-length ADAMTS13 variants for a single neutralizing antibody. Antibody escape rates for 7 neutralizing antibodies (Ab 4-16, ab4-20, ab60, ab61, ab64, ab65, and Ab 67) were measured in 12 full-length ADAMTS13 variants. Neutralizing antibody escape rates were calculated by comparing relative binding levels to WT ADAMTS13 values and substituting relative escape rates into the following equation using them: escape rate (%) = (binding capacity of 1-variant/binding capacity of WT ADAMTS 13) ×100.
Figure 5 shows the results of confirming the ability to escape a single neutralizing antibody in media expressing Fc-linked MDTCS fragment variants. The selected 12 variants were MDTCS fragmented and then Fc-linked variants were combined with the selected mutant amino acid residues and the neutralizing antibody escape rate and relative activity of 8 neutralizing antibodies (Ab 3-01, ab4-16, ab4-20, ab60, ab61, ab64, ab65, and Ab 67) of ADAMTS13 were measured using medium in which a total of 14 variants (including DM1 and DM2 variants, with two amino acid variations) were expressed. Escape rate (%) = (binding capacity of 1-variant/binding capacity of WT ADAMTS 13) ×100; relative activity (%) = specific potency of variant/specific potency of WT ADAMTS13 x 100.
Figure 6 shows the results of confirmation of the ability to escape mixed neutralizing antibodies under media conditions expressing Fc-linked MDTCS fragment variants. The 9 neutralizing antibodies (Ab 3-01, ab4-16, ab4-20, ab60, ab61, ab64, ab65, ab66, and Ab 67) mixed in equal proportions were mixed into and reacted with: 4nM medium which expresses MDTCS-Fc (i.e., control) and 12 candidate variants. The remaining activity of each candidate variant is then calculated by substitution into the following equation: residual activity (%) =a ≡b×100 (a: activity under mixed neutralizing antibody conditions, B: activity under conditions without neutralizing antibody treatment).
Fig. 7 shows the results of confirming the ability to escape a single neutralizing antibody under conditions of Fc-linked MDTCS fragment variant medium and purification solution. The neutralizing antibody escape rate and relative activity against 8 individual neutralizing antibodies (Ab 3-01, ab4-16, ab4-20, ab60, ab61, ab64, ab65 and Ab 67) were measured using media expressing 12 variants (1C 03, 2B01, 2B02, 3B05, 4E11, 4H07, 5G08, 7a02, 8D01, 8D05, DM1 and DM 2) or purified solutions purified using the Phytip system (fig. 7a and 7B). The concentration of the purified solution was determined by Fc ELISA. Escape rate (%) = (binding capacity of 1-variant/binding capacity of MDTCS-Fc) ×100, relative activity (%) = specific potency of variant/specific potency of MDTCS-fc×100. Separately, blue bars (bar) represent purified variant proteins, and grey bars represent median values of expressed variant proteins.
Fig. 8 shows the results of confirmation of the ability to escape mixed neutralizing antibodies under conditions of Fc-linked MDTCS fragment variant medium and purification solution. The 8 neutralizing antibodies (Ab 3-01, ab4-16, ab4-20, ab60, ab61, ab64, ab65, and Ab 67) mixed in equal proportions were mixed into the following and allowed to react: culture medium, which expresses MDTCS-Fc (i.e., control), and 12 candidate variants, or 4nM purified solution. The remaining activity of each candidate variant was then measured. The remaining activity was calculated by substituting into the following equation: residual activity (%) =a ≡b×100 (a: activity under mixed neutralizing antibody conditions, B: activity under conditions without neutralizing antibody treatment).
Fig. 9 shows a graph illustrating the pharmacokinetic results of Fc-linked MDTCS fragment variants. Plasma was obtained hourly after intravenous administration of MDTCS or Fc (IgG 1-YTE) linked MDTCS and four final candidate variant (1C 03, 5C09, 7a02 and DM 2) fragment proteins to the tail of the mice. The proteins are administered such that the specific titer of each substance may be 160IU/kg and the activity of the remaining substances in the plasma obtained per hour is measured by an activity assay.
FIG. 10 shows the results of confirmation of the ability to escape a single neutralizing antibody under conditions of IgG1-YTE linked MDTCS fragment variant medium. The five variants selected were MDTCS fragmented and the escape neutralization antibody rates and relative activities for eight individual neutralizing antibodies (Ab 3-01, ab4-16, ab4-20, ab60, ab61, ab64, ab65 and Ab 67) were measured using medium expressing IgG1-YTE linked variants (fig. 10a and 10 b). Escape rate (%) = (binding capacity of 1-variant/binding capacity of MDTCS-IgG 1-YTE) ×100; relative activity (%) = specific potency of variant/MDTCS-IgG 1-YTE x 100.
FIG. 11 shows the results of confirmation of the ability to escape mixed neutralizing antibodies under media conditions expressing IgG1-YTE linked MDTCS fragment variants. The 9 neutralizing antibodies (Ab 3-01, ab4-16, ab4-20, ab60, ab61, ab64, ab65, ab66, and Ab 67) mixed in equal proportions were mixed into and reacted with: 4nM medium which expresses MDTCS-IgG1-YTE (i.e., control), and 5 candidate variants. The remaining activity of each candidate variant was then calculated by the following equation: residual activity (%) =a ≡b×100 (a: activity under mixed neutralizing antibody conditions, B: activity under conditions without neutralizing antibody treatment).
FIG. 12 shows the results of confirmation of the ability to escape mixed neutralizing antibodies under IgG1-YTE linked MDTCS fragment variant medium conditions. The 9 neutralizing antibodies (Ab 3-01, ab4-16, ab4-20, ab60, ab61, ab64, ab65, ab66, and Ab 67) mixed in equal proportions were mixed into and reacted with: 4nM purified solution with MDTCS-IgG1-YTE (i.e., control), and 5 candidate variants. The remaining activity of each candidate variant was then calculated by the following equation: residual activity (%) =a ≡b×100 (a: activity under mixed neutralizing antibody conditions, B: activity under conditions without neutralizing antibody treatment).
Fig. 13 shows a schematic diagram illustrating the operation of using an aTTP-simulated mouse model for testing escape neutralization antibody rates (fig. 13 a) and a graph showing residual ADAMTS13 activity according to the dose of variant candidates (fig. 13 b), respectively.
Fig. 14 shows a schematic diagram illustrating test procedures for maintaining residual activity of ADAMTS13 and clinical symptoms according to concentration of DM2-IgG1-YTE, which shows the most excellent neutralizing antibody escape rate (fig. 14 a) of a model mouse simulated using aTTP, a schematic diagram illustrating platelet and LDH levels and improvement of ADAMTS13 activity (fig. 14 b) and observation of clinical symptoms (fig. 14 c), respectively.
Fig. 15 shows a schematic diagram illustrating the test procedure for the improvement of the presence/absence of hematological and clinical symptoms and the extent of recovery of human ADAMTS13 activity by administration of DM2-IgG1-YTE to cTTP mouse models (fig. 15 a), and observations of platelet and LDH levels and improvement of ADAMTS13 activity and recovery of ADAMTS13 activity, respectively (fig. 15 b).
Embodiments of the invention
Hereinafter, the present invention will be described in more detail by way of examples. These examples are merely for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.
Examples
Experimental method
Construction of human ADAMTS13 variant libraries by random mutagenesis
The following were prepared: an expression vector comprising human wild-type ADAMTS13, and two recipient vectors for cloning mutated MDTCS (metalloprotease, desmin-like, thrombospondin type 1 (TSP 1) repeat, cys-rich and spacer) or spacer (S) domain regions. Error-prone PCR (GeneMorph II random mutagenesis kit, agilent Technologies) was performed on the MDTCS or S domain region of ADAMTS13 using the oligonucleotides in table 1 below, and PCR products of amplified MDTCS or S domains were obtained by elution after agarose gel electrophoresis eluted by electrophoresis. After the eluted PCR product and the recipient vector were ligated using the Golden Gate cloning method, the resulting product was transformed into E.coli (E.coli) to prepare a library of variants produced by random mutagenesis. Plasmid DNA of transfected grade was extracted from 384 E.coli colonies per library using plasmid Plus 96Miniprep kit (QIAGEN) to obtain 768 mutant ADAMTS13 variants in total. The type and position of amino acid variation was confirmed by nucleotide sequence analysis of the corresponding DNA.
TABLE 1
Nucleotide sequence of primer for error-prone PCR
Transient expression of human ADAMTS13 variants
For expression of ADAMTS13 variant proteins, concentrations of 3X 10 were prepared by dilution in an Expi293 expression medium 6 Individual cells/mL of Expi293F TM Cells (Thermo Fisher, catalog number A14527). In a first tube, 2.5. Mu.g of plasmid DNA was diluted in opti-MEM I-reduced serum medium (Thermo Fisher, catalog No. 31985-070) to a final volume of 152.5. Mu.L, while in another tube 8. Mu.L of Expifectamine 293 and 140. Mu.L of opti-MEM I-reduced serum medium were mixed and allowed to stand for 5 minutes. 152.5. Mu.L of the diluted plasmid DNA was added to the mixed solution in the second tube, and allowed to stand for 20 minutes. 2.5mL of the prepared cells were aliquoted into each of 24 deep well plates. A mixture of the previously prepared plasmid DNA and the Expiectamine 293 was dispensed into cells and incubated at 37℃and CO 2 The culture was performed in an incubator under the conditions at 225 rpm. After 24 hours, 15. Mu.L of ExpiFectamine transfection enhancer 1 and 150. Mu.L of enhancer 2 were added thereto, and cultured for 5 days. On day 6, cultures were spun at 300rcf for 5 minutes to collect supernatant and the resulting products were used for analysis for selection of variants.
Construction of human and mouse ADAMTS13 neutralizing antibodies
To construct ADAMTS13 neutralizing antibodies that bind to different domains, full-length recombinant human ADAMTS13 (R&D Systems, catalog No. 6156-AD) as antigen and use of human combinatorial antibody librariesPhage library (HuCAL, bio-Rad)16 fabs with excellent binding capacity to ADAMTS13 were selected, and among them, six antibodies (Ab 60, ab61, ab64, ab65, ab66, and Ab 67) that bound to each of the MDTCS moiety and the C-terminal moiety were prepared as full-length antibodies. Ab3-01, ab 4-16 and Ab 4-20, which are known to specifically bind to the S domain in plasma of aTTP patients, were also constructed as full length antibodies and used for selection analysis of variants.
Measurement of expression concentration of human ADAMTS13 variants
To confirm the expression level of the ADAMTS13 variant protein present in the medium obtained by transient expression, ELISA was performed using an anti-ADAMTS 13 antibody (Ab 66,GC Green Cross antibody). 100. Mu.L of an anti-ADAMTS 13 antibody diluted to 2. Mu.g/mL in PBS (Lonza, 17-516Q) was dispensed into each well of a 96-well EIA/RIA high binding microplate (Corning, catalog No. 3590) and allowed to react for two hours at room temperature. The microplate was then washed three times with wash buffer (0.1 v/v% tween 20 in PBS). Blocking buffer (1 w/v% bovine serum albumin in PBS) was dispensed at 300. Mu.L/well and reacted for two hours at room temperature, followed by three washes with wash buffer. Recombinant human ADAMTS13 (R) &D Systems, 6156-AD) was used as a standard sample, and the culture to be measured was diluted 16-fold or 32-fold with a blocking buffer and dispensed at 100 μl/well and reacted by stirring at 500rpm for one hour at room temperature. After three washes, the anti-6 XHis tag antibody (Abcam, catalog No. Ab 1187) was diluted 1:10,000 and dispensed at 100. Mu.L/well, and then reacted by stirring at 500rpm for 1 hour at room temperature. After six washes, TMB (3, 3', 5' -tetramethylbenzidine) was dispensed at 100. Mu.L/well and allowed to react at room temperature for 10 minutes. Then, stop buffer (H) was dispensed at 100. Mu.L/well 2 SO 4 ) And absorbance was measured at 450nm using a microplate reader. After substituting the measured absorbance into the standard sample response curve and calculating the concentration, the dilution factor is calibrated to determine the final concentration.
Measurement of expression concentration of Fc fusion MDTCS fragments and variants
Goat anti-human IgG Fc (Abcam, catalog number A3803) diluted to 2. Mu.g/mL with PBS (Lonza, catalog number 17-516Q) was dispensed at 100. Mu.L/well to 96-well EIA/RIA high knotsMicroplates (Corning, cat No. 3590) were combined and allowed to react for two hours at room temperature. The microplate was then washed three times with wash buffer (0.1 v/v% tween 20 in PBS). Blocking buffer (1 w/v% bovine serum albumin in PBS) was dispensed at 300. Mu.L/well and allowed to react for two hours at room temperature, then washed three times with wash buffer. The Fc-fusion MDTCS fragment protein with a fixed concentration was used as a standard sample, and the sample to be measured was diluted with a blocking buffer to be included in the standard range. The resulting product was then dispensed at 100. Mu.L/well and reacted for one hour by stirring at 400rpm at room temperature. After three washes, anti-human IgG (Fc specific) peroxidase antibody (Sigma-aldrich, catalog number A0170) was diluted 1:10,000 and dispensed at 100. Mu.L/well and reacted for one hour by stirring at 400rpm at room temperature. After six washes, TMB was dispensed at 100. Mu.L/well and allowed to react for 30 minutes at room temperature. Stop buffer was then dispensed at 100. Mu.L/well (0.5M H 2 SO 4 ) And absorbance was measured at 450nm with a microplate reader.
Measurement of human ADAMTS13 variant Activity
To measure the activity of ADAMTS13 variants, experiments were performed using a manual of Technozyme ADAMTS activity kit (Technoclone, catalog No. 5450701). GST-vWF73 substrate was dispensed at 100. Mu.L/well, allowed to react for one hour at room temperature, and then washed three times with wash buffer. The calibrator in the kit was used as a standard sample, and the heat-inactivated plasma was used as a culture to be measured by adding a stock solution or a 3-fold dilution thereof at 100. Mu.L/well, and allowed to react at room temperature for 30 minutes. After three washes with wash buffer, the conjugate was added at 100 μl/well, allowed to react for one hour at room temperature, and washed three more times with wash buffer. TMB developer was added at 100. Mu.L/well and allowed to react for 30 minutes, and then 100. Mu.L of stop buffer was added to terminate the reaction, and absorbance was measured at 450nm with an ELISA reader. To calculate the specificity titers (IU/mg), the expression results (μg/mL) were applied to the activity values (IU/mL), and the relative activity (%) was calculated using the specificity titers of wild-type (WT) ADAMTS13 and variants by substitution into the following equation:
Relative activity (%) = specific potency of variant/specific potency of wild-type ADAMTS13 x 100
Confirmation of escape Rate of human ADAMTS13 neutralizing antibodies
To confirm the ability of each ADAMTS13 variant to escape ADAMTS13 neutralizing antibodies, ELISA was performed using eight anti-ADAMTS 13 antibodies (Ab 3-01, ab 4-16, ab4-20, ab60, ab61, ab64, ab65, and Ab 67,GC Green Cross). anti-ADAMTS 13 antibodies diluted to 1 to 2. Mu.g/mL with PBS (Lonza, cat. No. 17-516Q) were added to a 96-well EIA/RIA high binding microplate (Corning, cat. No. 3590) at 100. Mu.L/well and allowed to react for two hours at room temperature. The microplate was then washed three times with wash buffer (0.1% tween 20 in PBS). Blocking buffer (1% BSA/PBS) was added at 300. Mu.L/well and allowed to react for two hours at room temperature and then washed three times with wash buffer. Recombinant human ADAMTS13 (R)&D Systems, catalog No. 6156-AD) as standard sample, and the culture to be measured was diluted to 25 to 50ng/mL with a blocking buffer and dispensed at 100 μl/well and reacted by shaking at 500rpm at room temperature for one hour. After three washes, the anti-6 XHis-tagged antibody (Abcam, catalog No. Ab 1187) was diluted 1:10,000 and dispensed at 100. Mu.L/well and reacted for one hour by shaking at 500rpm at room temperature. After six washes, TMB was dispensed at 100. Mu.L/well and allowed to react at room temperature for 10 to 20 minutes. Then, stop buffer (H) was dispensed at 100. Mu.L/well 2 SO 4 ) And absorbance was measured at 450nm using a microplate reader. Neutralizing antibody escape rates were calculated such that relative binding levels were compared to wild-type ADAMTS13 values and then relative escape rates were substituted into the following equation using the same:
escape rate (%) = (binding ability of 1-variant/binding ability of wild-type ADAMTS 13) ×100
Construction of MDTCS and Fc (IgG 1-YTE) conjugated MDTCS variants and production of proteins
An expression vector was constructed in which IgG1-YTE (hinge-Fc) was conjugated to MDTCS or MDTCS variant fragments, and gene synthesis was performed (Thermo Fisher Scientific,gene synthesis) as shown in table 2 below. Expression vectors were constructed by inserting each synthetic gene in table 2 into a pMSID2 vector (constructed by GC Green Cross). At 3X 10 5 CHO DG44 (S) cells were subcultured per mL using CDM4CHO (GE, catalog No. SH 30557.02) medium according to the expected amount. On the day of transfection, live 3X 10 was collected 7 Individual cells, suspended in OptiPro TM In SFM (Gibco, catalog number 12309019), diluted to a final volume of 1X 10 7 Individual cells/mL (3 mL) and added to a 125mL shake flask and mixed with CO 2 Shaking incubator (37 ℃,5% CO) 2 ) Is cultured at 140 rpm. Using OptiPro TM SFM (tube 1) 30 μg of expression vector was mixed to a total volume of 1.5mL. 90. Mu.L of transporter 5 TM PEI (Polyscience, catalog number 26008-5) and 1,410. Mu.L of OptiPro TM SFM mix (tube 2). After the previously prepared tube 2 was added to tube 1 and gently mixed, the mixture was allowed to react at room temperature for 20 to 30 minutes. 3mL of the DNA-PEI complex was added drop-wise to 3mL of the CHO DG44 (S) cells previously prepared. The obtained product is treated with CO 2 Shaking incubator (37 ℃,5% CO) 2 ) After 4 hours, 24mL of CDM4CHO medium was added to 6mL of CHO DG44 (S)/DNA: PEI complex and incubated under the same conditions. After 48 hours of incubation, cell number and cell viability were measured and the resulting product was added to a centrifuge tube to a volume of 15×10 5 The individual cells were centrifuged at 1,200rpm for 5 minutes and the supernatant removed and suspended in 30mL of CDM4CHO medium containing 20nM MTX and then inoculated into shake flasks and cultured. Subculturing is performed every 3 to 4 days until the cell viability is restored to 90% or more. The final overexpressing cell line was cultured on a large scale and used for protein purification. For MDTCS protein purification, the prepared culture was centrifuged to remove cells, and then filtered using a sterile filter made of cellulose acetate (Sartobran P,5235307H7-OO, sartorius) after centrifuging the prepared culture solution. The filtered culture was concentrated and then purified using anion exchange chromatography (QSepharose Fast Flow, GE), multi-mode chromatography (hydroxyapatite, bio-Rad) and affinity chromatography The spectra (Blue Sepharose, GE) were purified and the MDTCS variant IgG1-YTE conjugated protein was filtered, such that cultures of the overexpressing cell lines were centrifuged to remove cells and subsequently purified using an antibacterial filter made of cellulose acetate (Sartobran P,5235307H7-OO, sartorius). The filtered culture was purified using affinity chromatography (protein a, mabSelect SuRe, GE Healthcare) and size exclusion chromatography. It was confirmed that the purified protein had a purity of 95% or more.
TABLE 2
Amino acid sequence of MDTCS-IgG1-YTE fusion
* Blackbody: MDTCS/underline: hinge/italics: fc (Fc)
Confirmation of pharmacokinetics of MDT CS-hinge-Fc fusion
To compare the duration of fusion of MDTCS-IgG1-YTE (hinge-Fc) in blood and pharmacokinetic parameters, 44 or 52C 57BL/6 mice (4 mice/each time of blood collection) were used for each group. After 160IU/kg of MDTCS (control group) and 3 substances (where IgG1-YTE was fused with MDTCS variants) (test group) were injected into tail vein, blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96 and 168 hours. 500. Mu.L of blood was collected from the heart by a syringe containing 10% trisodium citrate (i.e., an anticoagulant) and centrifuged at 2,000Xg for 20 minutes at 4℃to separate the supernatant, and thereby obtain plasma. The activity of MDTCS variant-hinge-Fc present in plasma was measured according to the manual of Technozyme ADAMTS activity kit (Technoclone, catalog No. 5450701). In addition, plasma obtained from 44 mice was assayed for activity to obtain an average (0.0772 IU/mL) of mouse ADAMTS13 activity in the mouse plasma. By removing the corresponding values from the activity measurements of PK study samples, mouse ADAMTS13 activity inherent in mouse plasma can be excluded and only the activity results of the administered substances obtained. The data were analyzed by non-compartmental analysis using a simple combination method and WINNONLIN. t is t 1/2 Representing a drugAUC of (a) blood half-life Inf Represents the area from 0 to infinity time under the activity profile, and the mean residence time (mean residence time, MRT) represents the mean residence time of the drug molecule in the body.
Confirmation of ADAMTS13 binding site of neutralizing antibody
The nucleic acid sequences of 6 ADAMTS13, in which the full-length wild-type or C-terminal domains were deleted sequentially, were cloned into pcdna 3.1V 5-His B vector (Invitrogen, catalog No. V81020). The constructed DNA was immunoprecipitated by adding protein A-Sepharose4B using the culture obtained by the above-described transient expression method, followed by Western blotting. Specifically, three 30nM ADAMTS13 neutralizing antibodies (Ab 4-16, ab66, and Ab 67) were added to normal control plasma (HemosIL, catalog number 00020003110). After the following co-addition, 500. Mu.L of binding buffer (50 mM Tris-HCl, pH 8.0, 150mM NaCl,1%Triton X-100 and 1% BSA) was added thereto and allowed to react at 4℃for 24 hours: 10mL of plasma comprising neutralizing antibodies, and a culture comprising 50ng of each ADAMTS13 fragment protein. After completion of the reaction, 20. Mu.L of protein A-Sepharose4B resin (Thermo Fisher Scientific, catalog No. 101041) was added thereto as a 50% slurry, and allowed to react at 4℃for 4 hours. The resin was collected by centrifugation at 845rcf for 1 min and then washed three times with wash buffer (50 mM Tris-HCl, pH 8.0, 150mM NaCl,1% Triton X-100). The resin bound protein was eluted by SDS-sample buffer (Novex, cat# NP 0007) containing 10% beta-mercaptoethanol and heated at 95℃for 5 min, followed by SDS-PAGE and anti-immunoblotting (by means of V5 antibody (Invitrogen, cat# R960-25)).
PoC evaluation of variants in a mouse model with aTTP mimetic disease
A mouse model with aTTP-mimicking disease was established by treating ADAMTS13KO mice with human ADAMTS13 neutralizing antibodies. ADAMTS13KO mice were prepared from Macrogen by using CRISPR/Cas9 system to induce large deletions and frameshift variations between exon 1 and exon 6 in ADAMTS13 gene. 9 ADAMTS13 neutralizing antibodies (Ab 3-01, ab4-16, ab4-20, ab60, ab61, ab64, ab65, ab66, and Ab 67) mixed in equal proportions were administered intravenously to 12 to 25 week old ADAMTS13KO mice at a dose of 0.54 mg/kg. After 15 minutes, the control substance (MDTCS-Fc) or five variants (1C 03, 2B02, 5C09, 7A02 and DM 2) were administered at a dose of 5,000IU/kg or 7,000IU/kg. Blood samples were collected 6 hours after administration to measure ADAMTS13 activity.
To confirm whether recovery of ADAMTS13 activity and hematological properties shown in aTTP model mice was improved by administration of DM2 variants, 9 ADAMTS13 neutralizing antibodies (Ab 3-01, ab4-16, ab4-20, ab60, ab61, ab64, ab65, ab66, and Ab 67) mixed in equal proportions were administered intravenously to 12 to 25 week old ADAMTS13KO mice at a dose of 0.54 mg/kg. After 15 minutes, the control substance (MDTCS-Fc) or variant DM2 was administered at a dose of 5,000IU/kg, 7,000IU/kg or 14,000IU/kg. After administration of 2,000iu/kg of recombinant human vWF (VEYVONDI) 30 minutes after administration of the neutralizing antibody, 700 μl of blood was collected 6 hours after this, and 300 μl of blood was added to EDTA tubes (BD Medical, REF 365974) and another 300 μl of blood was added to SST tubes (BD Medical, REF 365967), left at room temperature for 30 minutes, and then centrifuged at 3,000rpm for 15 minutes at 4 ℃ to separate serum. The remaining blood was mixed with sodium citrate at a ratio of 9:1 (blood: sodium citrate), and plasma was isolated and used to analyze the remaining activity of human ADAMTS13 in plasma. Whole blood in EDTA tubes was subjected to a general blood test such as Platelets (PLT) using a cytometer (ADVIA 2120i, siemens), while serum isolated in SST tubes was used for measurement of Lactate Dehydrogenase (LDH) using a blood biochemical analyzer (Hitachi, 7180). Statistical analysis used one-way ANOVA test and Tukey test.
PoC evaluation of variants in a mouse model with cTTP disease
An ADAMTS13 KO mouse was used to establish a mouse model with cTTP disease. ADAMTS13 KO mice of 12 to 25 weeks of age were dosed intravenously with vehicle or DM2 variants at doses of 20IU/kg, 60IU/kg, 180IU/kg and 360IU/kg, and after 15 minutes, recombinant human vWF (VEYVONDI) was dosed at 2000IU/kg, and blood samples were collected 6 hours after that. Whole blood was used for general blood tests (including measurement of platelet levels), while serum was used for measurement of LDH levels, and plasma was used for analysis of human ADAMTS13 activity.
Experimental results
Construction of human ADAMTS13 variants Using random mutagenesis
Regarding ADAMTS13 neutralizing antibodies possessed by aTTP patients, it is reported that the proportion of multiple domains of ADAMTS13 bind to MDTCS is relatively high, exceeding 97%, and that the S domain is the most prevalent binding site (Tersteg et al,2016; klaus et al, 2004; leken et al, 2005). Based on the above, each library was prepared by performing error-prone PCR such that a mutation targeting the MDTCS moiety or S domain could occur (fig. 1 a). As a result of obtaining DNA of 384 ADAMTS13 variants from each library and analyzing the nucleotide sequence, the total variation rate in the library of MDTCS fractions was 72.9%, and specifically, the silent mutation or non-mutation showed a rate of 23.7%, the empty (empty) vector or lack of sequence (pool sequence) was 3.1%, the nonsense mutation was 8.9%, the frameshift mutation was 4.9%, and the missense variation was 59.1% (fig. 1 b). For the S domain library, the overall mutation rate was 50.8%, and silent or non-mutated 44.5%, empty vector or absent sequence 4.7%, nonsense mutation 2.3%, shift mutation 3.1%, missense mutation 45.3% (fig. 1 b). Selection experiments were performed using about 500 variants that did not include those variants that exhibited silent or non-mutated, empty vector or lacking sequence, nonsense mutations, as described above.
Preparation of ADAMTS13 neutralizing antibodies and confirmation of binding sites
To confirm whether variants with mutations in the MDTCS region or S domain are able to escape neutralizing antibodies to ADAMTS13, fab was constructed using the HuCAL system (Bio-Rad) for antibodies recognizing different epitopes of human ADAMTS13 and 16 variants with excellent binding ability to human ADAMTS13 were selected. Ab 66 and Ab 67, which were confirmed to specifically bind to different domains in 16 variants, were generated as full length antibodies. Since Ab 4-16 is known to specifically bind to the S domain in the plasma of aTTP patients, it is produced in the form of full length antibodies (Casina et al, 2015), and the binding region of each antibody is as shown in fig. 2C, such that Ab 4-16 specifically binds to the S domain, ab 67 specifically binds to the MDTCS region, and Ab 66 specifically binds to the C-terminus (TSP-2 to TSP-8 domain).
Setting selection criteria for variants
Variants were evaluated by analyzing the relative results of non-mutated or silent mutant variants (hereinafter referred to as wild-type clones) having the same amino acid sequence as wild-type ADAMTS13 among the variants constructed by random mutagenesis. The activity and binding affinity of 59 wild-type clones for Ab 4-16 and Ab 67 were analyzed in total, and the results of the analysis of each wild-type clone were each represented by the results of the wild-type ADAMTS13 construct and are shown in fig. 3. As shown in fig. 3, escape rates and relative activities for Ab 4-16 and Ab 67 antibodies are shown for wild-type ADAMTS13 (59), mutant ADAMTS13 (304), and selected ADAMTS13 (26) containing conservative amino acid substitutions. Despite no variation, the wild-type clones showed relative differences in results from the wild-type ADAMTS13 constructs, and among the mutant ADAMTS13 (304), 26 ADAMTS13 mutants with escape rates or values greater than or equal to the relative activity were selected, as shown in table 3 below.
Specifically, for mutant ADAMTS13, the escape rate for Ab4-16 ranged from-29.5% to 33.4%, the escape rate for Ab67 ranged from-24.4% to 30.5%, and the relative activity showed a range of 62.7% to 128.9% (table 3). As a result of applying the normal distribution using the tri-sigma rule, the escape rate for Ab4-16 was-34.1% to 38.5%, the escape rate for Ab67 was-38.5% to 42.3%, and the range of relative activity was 47.0% to 145.2%, and therefore it was predicted that almost all WT clones would fall within the distribution. Thus, in selecting variants, the following selection criteria are applied: the escape rate for Ab4-16 was over 38.5%, the escape rate for Ab67 was over 42.3%, or the relative activity was 47.0% or higher (based on maximum 3 sigma).
TABLE 3
Escape rate and range of relative Activity against neutralizing antibody Ab4-16 or Ab67
Category(s) Escape Rate against Ab4-16 Escape Rate against Ab67 Relative Activity
Minimum value -29.5％ -24.4％ 62.7％
Maximum value 33.4％ 30.5％ 128.9％
Average value of 2.2％ 1.9％ 96.1％
Standard deviation of 12.1％ 13.5％ 16.4％
Mean-3 standard deviation -34.1％ -38.5％ 47.0％
Mean +3 standard deviation 38.5％ 42.3％ 145.2％
Selection of variants that escape ADAMTS13 neutralizing antibodies and have activity at levels greater than or equal to wild-type ADAMTS13
After transfection of WT clones and variants with amino acid variations into cells, the amount of protein present in the culture was measured and assays for binding affinity and activity for neutralizing antibodies were performed for 304 variants with expression concentrations of 50ng/mL or higher. Variants with amino acid variation showed a variety of distributions in binding affinity or relative activity against neutralizing antibodies compared to WT clones, and among them, 26 variants meeting selection criteria were finally selected (fig. 3a and 3 b). Of the 26 variants, 18 variants showed S domain variation, and in particular, 13 variants (1C 03, 1G07, 2B01, 2B02, 3B05, 3G04, 5C09, 5G08, 6B12, 7a02, 8C04, 8D01, and 8F 01) with S domain variation showed high escape rate against Ab4-16, and 5 variants (7E 01, 7G08, 8C02, 8D01, and 8D 05) showed characteristics with high relative activity (table 4). The 6 variants showed variation in the D domain and 5 of them (46, 3a06, 4C07, 4E11 and 4H 07) showed high escape rate characteristics against Ab 67. In addition, 6 variants with variation for the M domain, 2 variants with variation for the C domain and 2 variants with variation for the T domain were determined. Duplicate reproducibility of results was confirmed by three duplicate experiments on the 26 variants selected, and 12 variants that continued to maintain superiority over WT clones even in duplicate tests were selected as subjects for in vitro efficacy testing. Of the 12 variants, 1C03, 2B01, 2B02, 3B05, 5C09, 5G08, 7a02, and 8D01 were selected for their excellent Ab4-16 antibody escape rate, and 4C07, 4E11, and 4H07 were selected for their excellent escape rate against Ab67 antibody. Finally, 8D05 was chosen for its excellent relative activity.
To confirm the ability to escape additional neutralizing antibodies among the 12 selected variants, the inventors sought to confirm whether it was possible to even escape neutralizing antibodies Ab4-20, ab60, ab61, ab64, and Ab65 in addition to Ab4-16 and Ab67 antibodies used for screening. Of the 12 variants, 8 variants (except 8D 05) among the 9 variants having an amino acid variation in the S domain, i.e., 1C03, 2B01, 2B02, 3B05, 5C09, 5G08, 7a02, and 8D01 exhibited excellent escape rates against Ab4-16 and Ab4-20 prepared based on the sequences of ADAMTS13 autoantibodies possessed by the aTTP patient, and these variants also exhibited excellent escape abilities against Ab60 and Ab 61. In contrast, in the case of variants 4C07, 4E11 and 4H07 with amino acid variation in the D domain, the escape rate against Ab67 was excellent (fig. 4, table 5).
TABLE 4
Screening of 26 variants with excellent escape ability or relative Activity against Ab4-16 and Ab67 neutralizing antibodies
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TABLE 5
Confirmation of escape Capacity and relative Activity against Single neutralizing antibody in 12 selected variants
Confirmation of neutralizing antibody escape ability of Fc-linked MDTCS fragment variants
In one structure-function study, it was reported that, of the total 14 domains of ADAMTS13, the MDTCS domain (in which the CUB2 domain was removed from the C-terminal TSP-2) was shown to have a metalloprotease function similar to that of ADAMTS13 and thus to be able to cleave VWF (shellat et al 2005). This suggests that even only the MDTCS fragment, rather than the full-length ADAMTS13, can function as a therapeutic agent for TTP disease, and that it can escape neutralizing antibodies present in patient plasma in a truncated form that binds to the C-terminal portion.
In addition, the present inventors have attempted to further improve the structural stability of MDTCS fragments and extend half-life by linking Fc. The selected 12 variants were fragmented with MDTCS and Fc-linked variants were thus prepared, and DM1 and DM2 variants with two amino acid variations were additionally prepared by combining the selected variant amino acid residues. The final candidate substance is selected by measuring the escape rate and relative activity of the single or mixed neutralizing antibodies using the culture expressed in the cells by the above method and the purification solution. As a result of measuring the binding escape rate and relative activity against eight individual neutralizing antibodies using cultures, 2B01, 3B05, 4H07, 5G08, 8D05 and DM1 were shown to escape all neutralizing antibodies at similar levels (fig. 5, table 6). 1C03, 2B02, 5C09, 7a02, 8D01 and DM2 exhibited excellent escape rates for binding of 3-01 and Ab 60. All six candidates showed amino acid variation in the S domain and all except 8D01 had variation in amino acid 612. In the case of 4E11 and DM2, they were shown to have excellent escape rate of Ab67 binding, and both were shown to have variation in amino acid 314 of the D domain. In terms of relative activity, DM1 showed the lowest relative activity, 57.9%, and 1c03 showed the highest relative activity, 133.1%. The relative activity of all candidates was 78.2% to 125.1% with the exception of the two above, similar to the MDTCS-Fc control (Table 6). To confirm the escape ability of candidate variants other than 4C07 and 5C09, the MDTCS-Fc showed a remaining activity of 3.5% compared to the control MDTCS-Fc, and it was confirmed that 3B05, 4E11, 4H07, 5G08 and 8D05 had a remaining activity of 1.24% to 2.42% compared to the MDTCS-Fc by mixing each variant of the mixed neutralizing antibody of 7.5nM and the expression culture of 4nM under the condition that 9 neutralizing antibodies (Ab 3-01, ab4-16, ab4-20, ab60, ab61, ab64, ab65, ab66 and Ab 67) were mixed in the same ratio and reacting them at room temperature for one hour to measure the remaining activity, thus indicating a lower remaining activity compared to the MDTCS-Fc (fig. 6). In contrast, in the case of 1C03, 2B01, 2B02, 7a02, 8D01, DM1 and DM2, the residual activity remained at 7.69% to 18.81%, thus confirming that the escape ability for the mixed neutralizing antibody was excellent compared to MDTCS-Fc (fig. 6).
To confirm the neutralizing antibody escape capacity of candidate substance variants using the purification solution, protein purification was performed in 12 cultures except 4C07 and 5C09 using the Phytip system (protein a resin). The concentration of the purified solution was confirmed by Fc ELISA, and elution of the target protein was confirmed by silver staining. As a result of confirming the escape rate of the single neutralizing antibody using the purification solution, it was confirmed that most variants had a similar tendency to the result of evaluation in culture (fig. 7). The 2B01 5g08, 8D05 and DM1 variants showed escape neutralization antibody rates of 24.6% or higher for all eight neutralizing antibodies, and 1C03, 2B02, 7a02, 8D01 and DM2 showed excellent escape rates with 3-01 and Ab60 binding similar to the culture results. In the case of 4E11 and DM2, their excellent escape rate of binding to Ab67 was confirmed as with the culture results. The relative activity of each variant is shown in figure 7 and table 7. As a result of measuring the residual activity under the mixed neutralizing antibody conditions, it was confirmed that the MDTCS-Fc maintained the residual activity of 3.76%, and it was confirmed that the 3B05, 4E11, 4H07 and 8D05 variants showed the residual activity of 2.05% to 3.50%, thus showing higher inhibition as compared to the MDTCS-Fc. In contrast, in the case of 1C03, 2B01, 2B02, 5G08, 7a02, 8D01, DM1, and DM2, the remaining activities maintained were confirmed to be 6.34% to 12.8%, thus confirming that there was an excellent ability to escape neutralizing antibodies to the mixed neutralizing antibodies as compared with MDTCS-Fc (fig. 8).
Based on the above results, considering comprehensively the relative activities or escape rates for the mixed neutralizing antibodies, 1C03, 2B02, 7a02, DM2 and 5C09 were predicted to have excellent ability to escape the mixed neutralizing antibodies due to variation of amino acid 612, which were selected as five candidates for further study.
TABLE 6
Confirmation of the Single neutralizing antibody escape Capacity and relative Activity of MDTCS fragments comprising 14 variants (Medium Condition)
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TABLE 7
Confirmation of the Single neutralizing antibody escape Capacity and relative Activity of MDTCS fragments comprising 14 variants (purification solution conditions)
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The escape binding capacity and relative activity of eight individual neutralizing antibodies was measured in cell-expressed cultures by constructing DNA in which IgG1-YTE was conjugated to the last five MDTCS variant fragments selected. As a result, 1C03, 2B02, 5C09, and 7a02 showed excellent escape rates of binding to Ab3-01 and Ab60, and in the case of DM2, it was confirmed that the escape rates of binding to Ab3-01, ab60, and Ab67 were excellent (fig. 10). The relative activity was 98.6% to 127.7%, which is similar to that of MDTCS-IgG1-YTE at (FIG. 10). To confirm the escape ability of 5 variant substances compared to the control MDTCS-IgG1-YTE under the condition that 9 neutralizing antibodies were mixed in the same ratio, the mixed neutralizing antibodies (nine) were mixed with each variant of the expression culture, allowed to react at room temperature for one hour, and the remaining activity was measured. In the case of MDTCS-IgG1-YTE, the residual activity was maintained at 5.5%, and in the case of the five substances, the residual activity was maintained at 8.18% to 13.42%, thus confirming that the escape ability to neutralizing antibodies was excellent as compared with the control group (FIG. 11).
Proteins were purified from expression cultures in which IgG1-YTE was conjugated to the five MDTCS variant fragments described above using the Phytip system (protein a resin), and the residual activity and specific titers of the pooled neutralizing antibodies were measured in the purification solution. The concentration of the corresponding purified solution was confirmed by Fc ELISA, and elution of the target protein was confirmed by silver staining. As a result of measuring the residual activity under the mixed neutralizing antibody conditions, it was confirmed that MDTCS-IgG1-YTE was maintained at 0.4% and the residual activities of 1C03, 2B02, 5C09, 7A02, DM2 were maintained at 5.7%, 1.7%, 8.8%, 2.6%, 7.9%, respectively, thus confirming that the mixed antibody escape ability was superior to MDTCS-IgG1-YTE. As a result of measurement of the specific titers, it was confirmed that the four variants showed similar specific titers 19,091IU/mg to 22,379IU/mg (fig. 12) to the control group (18,030IU/mg) except for 7a02 (10,288IU/mg). As a result, it was confirmed that IgG1-YTE conjugated substances of the five variant fragments had excellent escape ability to the mixed neutralizing antibody, compared to the control group, and that the four variants other than 7a02 had similar specific titers to the control group.
Evaluation of whether half-life of MDTCS fragment variants was increased due to Fc ligation
To determine if Fc linked to MDTCS (IgG 1-YTE) would actually increase half-life, pharmacokinetic analysis was performed in mice. For this purpose, mice were administered MDTCS or MDTCS linked to IgG1-YTE and four final candidates (1C 03, 5C09, 7a02 and DM 2) variant fragment proteins at the tail vein, and plasma was then obtained at each set time. These substances were administered at 160IU/kg based on the specific titer of each substance, and the activity of the remaining substances in the plasma obtained at each set time was measured by an activity assay. The results obtained were summarized by a simple pooling method and pharmacokinetic analysis was performed using a non-compartmental analysis method. The half-life of MDTCS was measured as 2.898 hours, MDTCS-IgG1-YTE was 11.51 hours, and each variant exhibited half-life of 5.184 to 9.902 hours. By IgG1-YTE conjugation, half-life was prolonged by 1.79 to 3.97 fold compared to control MDTCS. In addition, the Mean Residence Time (MRT) value indicated that the mean residence time of the material conjugated by IgG1-YTE was 7.189 to 11.67 hours, thus indicating an increase in residence time compared to 3.743 hours (Table 8, FIG. 9). As a result, it was confirmed that IgG1-YTE fusion was effective in maintaining activity by blood half-life and average duration in vivo.
TABLE 8
Pharmacokinetic results
As described above, the present inventors have discovered 26 new variants that escape ADAMTS13 neutralizing antibodies that bind to the MDTCS region or S domain, or that exhibit activity greater than or equal to wild-type ADAMTS 13. Among them, 12 neutralizing antibodies having the most excellent escape ability or significantly excellent comparative activity against nine neutralizing antibodies were selected, and then MDTCS fragments, which are essential for vWF cleavage while being able to effectively escape the neutralizing antibodies bound to the C-terminus, were constructed, thereby determining variants having significantly improved rates of escape of autoantibodies bound to D, C or S domain. By IgG1-YTE conjugation, the variants of the invention can be effectively used as effective pharmacological ingredients with improved stability and persistence of physiological activity by increasing blood half-life.
Confirmation of variant PoC in an atttp-mimicking disease mouse model
To select the final candidate substance from the established atttp simulated mouse model, a control substance (MDTCS-IgG 1-YTE) or five variant candidate substances selected (1C 03-IgG1-YTE, 2B02-IgG1-YTE, 5C09-IgG1-YTE, 7a02-IgG1-YTE and DM2-IgG 1-YTE) were administered and the escape neutralizing antibody rates were evaluated (fig. 13 a). Of the five candidate variant substances, DM2-IgG1-YTE showed the highest residual activity of human ADAMTS13 at both doses of 5,000IU/kg and 7,000IU/kg (FIG. 13 b). DM2-IgG1-YTE substances that showed the best neutralizing antibody escape rate were finally selected and administered at various concentrations to confirm the ability to maintain residual activity of human ADAMTS13 and the extent of clinical symptom relief (fig. 14 a); as a result, it was confirmed that the degree of remission increased with the increase in DM2 dose, and it was observed that the improvement in platelet and LDH levels was relatively high by DM2-IgG1-YTE administration compared to MDTCS-IgG1-YTE administration in 7,000IU/kg administration group (fig. 14 b). Consistent with the improvement in platelet and LDH levels, an increase in residual activity proportional to the administered dose of the control or candidate substance was observed as a result of the human ADAMTS13 activity test (fig. 14 b). As a result of observation of clinical symptoms, it was confirmed that mortality or hematuria shown in the aTTP simulated mouse model was reduced by administration of the control substance or the candidate substance. In particular, in the case of the DM2-IgG1-YTE treated group, no death was observed at any dose, and no subject having hematuria symptoms was observed when administered at a dose of 7,000IU/kg or higher (fig. 14 c). The average residual activity of DM2-IgG1-YTE was 0.32IU/mL, 9.6 times that of MDTCS-IgG1-YTE when administered at a dose of 7,000IU/kg, and it was determined that the difference in clinical symptom observations was due to such a difference in residual activity. As a result of general blood tests, clinical chemistry tests, and observation of clinical symptoms, it was confirmed that administration of MDTCS-IgG1-YTE and DM2-IgG1-YTE tended to improve clinical symptoms of aTTP in an aTTP-simulated mouse model, and PoC could be confirmed in vivo in this way. In particular, it was confirmed that DM2-IgG1-YTE administration showed excellent improvement when administered at a dose of 7,000IU/kg, as compared to MDTCS-IgG1-YTE administration.
confirmation of variant PoC in cTTP disease mouse model
The presence of improvement in the hematological and clinical symptoms of TTP disease present in the cTTP mouse model and the extent of recovery of human ADAMTS13 activity were confirmed by using DM2-IgG1-YTE, which showed the most excellent effect among the five candidate variants of the aTTP-simulated mouse model (fig. 15 a). It was confirmed that, according to the increase in DM2-IgG1-YTE dose, the increase in platelet and LDH levels occurred in a concentration-dependent manner, and that significant increase in platelets was observed in the group to which DM2-IgG1-YTE was administered at 180, 360IU/kg compared to the control group, and that recovery comparable to the normal level was shown particularly in the case of the group to which DM2-IgG1-YTE was administered at 360IU/kg (fig. 15 b). Regarding the LDH level, it was confirmed that LDH was recovered to a level similar to that of the control group in the group administered at 180IU/kg (fig. 15 b). In terms of ADAMTS13 activity, the average activity was shown to be 0.1IU/mL or higher starting from the group to which DM2-IgG1-YTE was administered at a dose of 60IU/kg, and in the group to which DM2-IgG1-YTE was administered at a dose of 360IU/kg, the average activity was measured to be 1.08IU/mL (FIG. 15 b). Thus, DM2-IgG1-YTE variants were found to be effective in ameliorating the clinical symptoms of cTTP-disease mice and restoring ADAMTS13 activity.
Since certain specific portions of the invention have been described in detail above, it will be apparent to those of ordinary skill in the art that these specific descriptions are merely of preferred embodiments, and it will be clear that the scope of the invention is not limited thereto. Accordingly, the true scope of the invention should be defined by the following claims and their equivalents.
Reference to the literature
1.Miesbach W，Menne J，Bommer M，Schonermarck U，Feldkamp T，Nitschke M，Westhoff TH，Seibert FS，Woitas R，Sousa R，Wolf M，Walzer S，Schwander B.Orphanet Journal of Rare Diseases(2019)14：260.doi：10.1186/s13023-019-1240-0.
2.Ercig B，Wichapong K，Reutelingsperger CPM，Vanhoorelbeke K，Voorberg J，Nicolaes GAF.Thromb Haemost.2018；118(1)：28-41.doi：10.1160/TH17-06-0404
3.Johanna A Kremer Hoving，Paul Coppo，Bernhard Lammle，Joel L Moake，Toshiyuki Miyata，Karen Vanhoorelbeke.Nat Rev Dis Primers.2017Apr 6；3：17020.doi：10.1038/nrdp.2017.20.
4.Genetic and Rare Diseases Information Center(GARD)-an NCATS Program.10October 2018
5.Tersteeg C，Verhenne S，Roose E，et al.Expert Rev Hematol.2016；9(2)：209-221.doi：10.1586/17474086.2016.1122515
6.Klaus C，Plaimauer B，Studt JD，et al.Blood.2004；103(12)：4514-4519.doi：10.1182/blood-2003-12-4165
7.Luken BM，Turenhout EA，Hulstein JJ，Van Mourik JA，Fijnheer R，Voorberg J.Thromb Haemost.2005；93(2)：267-274.doi：10.1160/TH04-05-0301
8.Veronica C.Casina，Wenbing Hu，Jian-Hua Mao，Rui-Nan Lu，Hayley A.Hanby，Brandy Pickens，Zhong-Yuan Kan，Woon K.Lim，Leland Mayne，Eric M.Ostertag，Stephen Kacir，Don L.Siegel，S.Walter Englander，and X.Long Zheng.Proc Natl Acad Sci U S A.2015Aug 4；112(31)：9620-5.doi：10.1073/pnas.1512561112.
9.Suresh G Shelat，Jihui Ai，and X.Long Zheng.Semin Thromb Hemost.2005Dec；31(6)：659-72.doi：10.1055/s-2005-925472.
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Arg Gly Val Thr Gln Leu Gly Gly Ala Cys Ser Pro Thr Trp Ser Cys
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Leu Ile Thr Glu Asp Thr Gly Phe Asp Leu Gly Val Thr Ile Ala His
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Glu Ile Gly His Ser Phe Gly Leu Glu His Asp Gly Ala Pro Gly Ser
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Gly Cys Gly Pro Ser Gly His Val Met Ala Ser Asp Gly Ala Ala Pro
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Arg Ala Gly Leu Ala Trp Ser Pro Cys Ser Arg Arg Gln Leu Leu Ser
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Leu Leu Ser Ala Gly Arg Ala Arg Cys Val Trp Asp Pro Pro Arg Pro
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Gln Pro Gly Ser Ala Gly His Pro Pro Asp Ala Gln Pro Gly Leu Tyr
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Tyr Ser Ala Asn Glu Gln Cys Arg Val Ala Phe Gly Pro Lys Ala Val
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Ala Cys Thr Phe Ala Arg Glu His Leu Asp Met Cys Gln Ala Leu Ser
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Cys His Thr Asp Pro Leu Asp Gln Ser Ser Cys Ser Arg Leu Leu Val
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Pro Leu Leu Asp Gly Thr Glu Cys Gly Val Glu Lys Trp Cys Ser Lys
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Gly Gly Gly Val Val Thr Arg Arg Arg Gln Cys Asn Asn Pro Arg Pro
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Ala Phe Gly Gly Arg Ala Cys Val Gly Ala Asp Leu Gln Ala Glu Met
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Cys Asn Thr Gln Ala Cys Glu Lys Thr Gln Leu Glu Phe Met Ser Gln
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Gln Cys Ala Arg Thr Asp Gly Gln Pro Leu Arg Ser Ser Pro Gly Gly
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Ala Ser Phe Tyr His Trp Gly Ala Ala Val Pro His Ser Gln Gly Asp
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Ala Leu Cys Arg His Met Cys Arg Ala Ile Gly Glu Ser Phe Ile Met
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Lys Arg Gly Asp Ser Phe Leu Asp Gly Thr Arg Cys Met Pro Ser Gly
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Pro Arg Glu Asp Gly Thr Leu Ser Leu Cys Val Ser Gly Ser Cys Arg
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Thr Phe Gly Cys Asp Gly Arg Met Asp Ser Gln Gln Val Trp Asp Arg
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Cys Gln Val Cys Gly Gly Asp Asn Ser Thr Cys Ser Pro Arg Lys Gly
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Ser Phe Thr Ala Gly Arg Ala Arg Glu Tyr Val Thr Phe Leu Thr Val
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Thr Pro Asn Leu Thr Ser Val Tyr Ile Ala Asn His Arg Pro Leu Phe
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Thr His Leu Ala Val Arg Ile Gly Gly Arg Tyr Val Val Ala Gly Lys
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Met Ser Ile Ser Pro Asn Thr Thr Tyr Pro Ser Leu Leu Glu Asp Gly
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Glu Glu Ile Arg Ile Trp Gly Pro Leu Gln Glu Asp Ala Asp Ile Gln
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Val Tyr Arg Arg Tyr Gly Glu Glu Tyr Gly Asn Leu Thr Arg Pro Asp
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Ile Thr Phe Thr Tyr Phe Gln Pro Lys Pro Arg Gln Ala Trp Val Trp
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Ala Ala Val Arg Gly Pro Cys Ser Val Ser Cys Gly Ala Gly Leu Arg
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Trp Val Asn Tyr Ser Cys Leu Asp Gln Ala Arg Lys Glu Leu Val Glu
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Thr Val Gln Cys Gln Gly Ser Gln Gln Pro Pro Ala Trp Pro Glu Ala
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Cys Val Leu Glu Pro Cys Pro Pro Tyr Trp Ala Val Gly Asp Phe Gly
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Pro Gln Pro Cys Pro Ala Arg Trp Glu Val Ser Glu Pro Ser Ser Cys
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Thr Ser Ala Gly Gly Ala Gly Leu Ala Leu Glu Asn Glu Thr Cys Val
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Pro Gly Ala Asp Gly Leu Glu Ala Pro Val Thr Glu Gly Pro Gly Ser
835 840 845
Val Asp Glu Lys Leu Pro Ala Pro Glu Pro Cys Val Gly Met Ser Cys
850 855 860
Pro Pro Gly Trp Gly His Leu Asp Ala Thr Ser Ala Gly Glu Lys Ala
865 870 875 880
Pro Ser Pro Trp Gly Ser Ile Arg Thr Gly Ala Gln Ala Ala His Val
885 890 895
Trp Thr Pro Ala Ala Gly Ser Cys Ser Val Ser Cys Gly Arg Gly Leu
900 905 910
Met Glu Leu Arg Phe Leu Cys Met Asp Ser Ala Leu Arg Val Pro Val
915 920 925
Gln Glu Glu Leu Cys Gly Leu Ala Ser Lys Pro Gly Ser Arg Arg Glu
930 935 940
Val Cys Gln Ala Val Pro Cys Pro Ala Arg Trp Gln Tyr Lys Leu Ala
945 950 955 960
Ala Cys Ser Val Ser Cys Gly Arg Gly Val Val Arg Arg Ile Leu Tyr
965 970 975
Cys Ala Arg Ala His Gly Glu Asp Asp Gly Glu Glu Ile Leu Leu Asp
980 985 990
Thr Gln Cys Gln Gly Leu Pro Arg Pro Glu Pro Gln Glu Ala Cys Ser
995 1000 1005
Leu Glu Pro Cys Pro Pro Arg Trp Lys Val Met Ser Leu Gly Pro Cys
1010 1015 1020
Ser Ala Ser Cys Gly Leu Gly Thr Ala Arg Arg Ser Val Ala Cys Val
1025 1030 1035 1040
Gln Leu Asp Gln Gly Gln Asp Val Glu Val Asp Glu Ala Ala Cys Ala
1045 1050 1055
Ala Leu Val Arg Pro Glu Ala Ser Val Pro Cys Leu Ile Ala Asp Cys
1060 1065 1070
Thr Tyr Arg Trp His Val Gly Thr Trp Met Glu Cys Ser Val Ser Cys
1075 1080 1085
Gly Asp Gly Ile Gln Arg Arg Arg Asp Thr Cys Leu Gly Pro Gln Ala
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Gln Ala Pro Val Pro Ala Asp Phe Cys Gln His Leu Pro Lys Pro Val
1105 1110 1115 1120
Thr Val Arg Gly Cys Trp Ala Gly Pro Cys Val Gly Gln Gly Thr Pro
1125 1130 1135
Ser Leu Val Pro His Glu Glu Ala Ala Ala Pro Gly Arg Thr Thr Ala
1140 1145 1150
Thr Pro Ala Gly Ala Ser Leu Glu Trp Ser Gln Ala Arg Gly Leu Leu
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Phe Ser Pro Ala Pro Gln Pro Arg Arg Leu Leu Pro Gly Pro Gln Glu
1170 1175 1180
Asn Ser Val Gln Ser Ser Ala Cys Gly Arg Gln His Leu Glu Pro Thr
1185 1190 1195 1200
Gly Thr Ile Asp Met Arg Gly Pro Gly Gln Ala Asp Cys Ala Val Ala
1205 1210 1215
Ile Gly Arg Pro Leu Gly Glu Val Val Thr Leu Arg Val Leu Glu Ser
1220 1225 1230
Ser Leu Asn Cys Ser Ala Gly Asp Met Leu Leu Leu Trp Gly Arg Leu
1235 1240 1245
Thr Trp Arg Lys Met Cys Arg Lys Leu Leu Asp Met Thr Phe Ser Ser
1250 1255 1260
Lys Thr Asn Thr Leu Val Val Arg Gln Arg Cys Gly Arg Pro Gly Gly
1265 1270 1275 1280
Gly Val Leu Leu Arg Tyr Gly Ser Gln Leu Ala Pro Glu Thr Phe Tyr
1285 1290 1295
Arg Glu Cys Asp Met Gln Leu Phe Gly Pro Trp Gly Glu Ile Val Ser
1300 1305 1310
Pro Ser Leu Ser Pro Ala Thr Ser Asn Ala Gly Gly Cys Arg Leu Phe
1315 1320 1325
Ile Asn Val Ala Pro His Ala Arg Ile Ala Ile His Ala Leu Ala Thr
1330 1335 1340
Asn Met Gly Ala Gly Thr Glu Gly Ala Asn Ala Ser Tyr Ile Leu Ile
1345 1350 1355 1360
Arg Asp Thr His Ser Leu Arg Thr Thr Ala Phe His Gly Gln Gln Val
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Leu Tyr Trp Glu Ser Glu Ser Ser Gln Ala Glu Met Glu Phe Ser Glu
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Gly Phe Leu Lys Ala Gln Ala Ser Leu Arg Gly Gln Tyr Trp Thr Leu
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Gln Ser Trp Val Pro Glu Met Gln Asp Pro Gln Ser Trp Lys Gly Lys
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Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
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Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
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Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
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Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
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Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
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Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
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Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
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Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
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Gly Ile Leu Ala Cys Gly Phe Leu Leu Gly Cys Trp Gly Pro Ser His
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Phe Gln Gln Ser Cys Leu Gln Ala Leu Glu Pro Gln Ala Val Ser Ser
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Tyr Leu Ser Pro Gly Ala Pro Leu Lys Gly Arg Pro Pro Ser Pro Gly
50 55 60
Phe Gln Arg Gln Arg Gln Arg Gln Arg Arg Ala Ala Gly Gly Ile Leu
65 70 75 80
His Leu Glu Leu Leu Val Ala Val Gly Pro Asp Val Phe Gln Ala His
85 90 95
Gln Glu Asp Thr Glu Arg Tyr Val Leu Thr Asn Leu Asn Ile Gly Ala
100 105 110
Glu Leu Leu Arg Asp Pro Ser Leu Gly Ala Gln Phe Arg Val His Leu
115 120 125
Val Lys Met Val Ile Leu Thr Glu Pro Glu Gly Ala Pro Asn Ile Thr
130 135 140
Ala Asn Leu Thr Ser Ser Leu Leu Ser Val Cys Gly Trp Ser Gln Thr
145 150 155 160
Ile Asn Pro Glu Asp Asp Thr Asp Pro Gly His Ala Asp Leu Val Leu
165 170 175
Tyr Ile Thr Arg Phe Asp Leu Glu Leu Pro Asp Gly Asn Arg Gln Val
180 185 190
Arg Gly Val Thr Gln Leu Gly Gly Ala Cys Ser Pro Thr Trp Ser Cys
195 200 205
Leu Ile Thr Glu Asp Thr Gly Phe Asp Leu Gly Val Thr Ile Ala His
210 215 220
Glu Ile Gly His Ser Phe Gly Leu Glu His Asp Gly Ala Pro Gly Ser
225 230 235 240
Gly Cys Gly Pro Ser Gly His Val Met Ala Ser Asp Gly Ala Ala Pro
245 250 255
Arg Ala Gly Leu Ala Trp Ser Pro Cys Ser Arg Arg Gln Leu Leu Ser
260 265 270
Leu Leu Ser Ala Gly Arg Ala Arg Cys Val Trp Asp Pro Pro Arg Pro
275 280 285
Gln Pro Gly Ser Ala Gly His Pro Pro Asp Ala Gln Pro Gly Leu Tyr
290 295 300
Tyr Ser Ala Asn Glu Gln Cys Arg Val Ala Phe Gly Pro Lys Ala Val
305 310 315 320
Ala Cys Thr Phe Ala Arg Glu His Leu Asp Met Cys Gln Ala Leu Ser
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Cys His Thr Asp Pro Leu Asp Gln Ser Ser Cys Ser Arg Leu Leu Val
340 345 350
Pro Leu Leu Asp Gly Thr Glu Cys Gly Val Glu Lys Trp Cys Ser Lys
355 360 365
Gly Arg Cys Arg Ser Leu Val Glu Leu Thr Pro Ile Ala Ala Val His
370 375 380
Gly Arg Trp Ser Ser Trp Gly Pro Arg Ser Pro Cys Ser Arg Ser Cys
385 390 395 400
Gly Gly Gly Val Val Thr Arg Arg Arg Gln Cys Asn Asn Pro Arg Pro
405 410 415
Ala Phe Gly Gly Arg Ala Cys Val Gly Ala Asp Leu Gln Ala Glu Met
420 425 430
Cys Asn Thr Gln Ala Cys Glu Lys Thr Gln Leu Glu Phe Met Ser Gln
435 440 445
Gln Cys Ala Arg Thr Asp Gly Gln Pro Leu Arg Ser Ser Pro Gly Gly
450 455 460
Ala Ser Phe Tyr His Trp Gly Ala Ala Val Pro His Ser Gln Gly Asp
465 470 475 480
Ala Leu Cys Arg His Met Cys Arg Ala Ile Gly Glu Ser Phe Ile Met
485 490 495
Lys Arg Gly Asp Ser Phe Leu Asp Gly Thr Arg Cys Met Pro Ser Gly
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Pro Arg Glu Asp Gly Thr Leu Ser Leu Cys Val Ser Gly Ser Cys Arg
515 520 525
Thr Phe Gly Cys Asp Gly Arg Met Asp Ser Gln Gln Val Trp Asp Arg
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Cys Gln Val Cys Gly Gly Asp Asn Ser Thr Cys Ser Pro Arg Lys Gly
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Ser Phe Thr Ala Gly Arg Ala Arg Glu Tyr Val Thr Phe Leu Thr Val
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Thr Pro Asn Leu Thr Ser Val Tyr Ile Ala Asn His Arg Pro Leu Phe
580 585 590
Thr His Leu Ala Val Arg Ile Gly Gly Arg Tyr Val Val Ala Gly Lys
595 600 605
Met Ser Ile Ser Pro Asn Thr Thr Tyr Pro Ser Leu Leu Glu Asp Gly
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Arg Val Glu Tyr Arg Val Ala Leu Thr Glu Asp Arg Leu Pro Arg Leu
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Glu Glu Ile Arg Ile Trp Gly Pro Leu Gln Glu Asp Ala Asp Ile Gln
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Val Tyr Arg Arg Tyr Gly Glu Glu Tyr Gly Asn Leu Thr Arg Pro Asp
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Ile Thr Phe Thr Tyr Phe Gln Pro Lys Pro Arg Gln Ala Glu Pro Lys
675 680 685
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe
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Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
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Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
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Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
850 855 860
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
865 870 875 880
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
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Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
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Leu Ser Leu Gly Lys
915

Claims (13)

  1. Adamts13 (thrombospondin type 1 motif-containing desmin and metalloprotease, member 13) variant protein or functional fragment thereof comprising a substitution of one or more amino acid residues selected from the group consisting of: residue 85, residue 93, residue 126, residue 135, residue 278, residue 282, residue 308, residue 314, residue 317, residue 334, residue 364, residue 376, residue 413, residue 427, residue 452, residue 465, residue 567, residue 578, residue 585, residue 589, residue 607, residue 608, residue 609, residue 612, residue 618, residue 630, residue 635, residue 643, residue 651, residue 655, residue 656, residue 655, and residue 655 of SEQ ID NO.
  2. 2. The ADAMTS13 variant protein or functional fragment thereof of claim 1, wherein the ADAMTS13 variant protein is selected from each variant protein comprising amino acid residue substitutions at:
    -residues 85 and 317; residue 612; two or more of residue 282, residue 465 and residue 672; residue 635; residues 452 and 612; two or more of residues 278, 334 and 427; residue 618; residue 135; two or more of residues 126, 567 and 651; residue 413; residue 334; residue 314; two or more of residues 93, 364 and 376; residue 308; residue 656; residue 607; residues 612 and 624; residue 589; residues 650 and 656; residues 643; residue 585 and residue 658; two or more of residues 630, 654 and 664; four or more of residue 589, residue 608, residue 609, residue 624 and residue 655; residue 578; residue 585; residues 314 and 635; and residues 314 and 612.
  3. 3. The ADAMTS13 variant protein or functional fragment thereof of claim 1, wherein the ADAMTS13 variant protein comprises: residue 85 is replaced with Phe, residue 93 is replaced with Val, residue 126 is replaced with Met, residue 135 is replaced with Ile, residue 278 is replaced with Ile, residue 282 is replaced with Ala, residue 308 is replaced with Lys, residue 314 is replaced with Thr, residue 317 is replaced with His, residue 334 is replaced with Thr or Val, residue 364 is replaced with Arg, residue 376 is replaced with Asp, residue 413 is replaced with Asp, residue 427 is replaced with Asn, residue 452 is replaced with Ile, residue 465 is replaced with Asp, residue 567 is replaced with Ser, residue 334 is replaced with Leu, residue 585 is replaced with Asn or Met, residue 589 is replaced with Gln, residue 607 is replaced with Arg, residue 608 is replaced with Met, residue 609 is replaced with Leu, residue 612 is replaced with Phe or Tyr, residue 618 is replaced with Ser, residue 624 is replaced with Asp or Cys, residue 630 is replaced with Leu, residue 635 is replaced with Val, residue 643 is replaced with Phe, residue 650 is replaced with His, residue 651 is replaced with Asp, residue 654 is replaced with Gly, residue 655 is replaced with Val, residue 656 is replaced with Arg or His, residue 658 is replaced with His, residue 664 is replaced with Asn, and residue 672 is replaced with Val.
  4. 4. A fusion protein comprising:
    (a) The ADAMTS13 variant protein or functional fragment thereof of any one of claims 1-3; and
    (b) An Fc region of an IgG4 immunoglobulin conjugated to (a) above.
  5. 5. The fusion protein of claim 4, wherein the Fc region comprises a substitution of one or more amino acid residues selected from the group consisting of residue 22, residue 24, and residue 26 of SEQ ID No. 2.
  6. 6. The fusion protein of claim 5, wherein residue 22 is replaced with Tyr, residue 24 is replaced with Thr, and residue 26 is replaced with Glu, respectively.
  7. 7. The fusion protein of claim 5, further comprising a hinge region of an IgG1 immunoglobulin between (a) and (b).
  8. 8. The nucleotide encoding: an ADAMTS13 variant protein or functional fragment thereof according to any one of claims 1-3; or a fusion protein according to any one of claims 4 to 7.
  9. 9. A composition for preventing or treating thrombotic diseases, comprising as active ingredients: an ADAMTS13 variant protein or functional fragment thereof according to any one of claims 1-3; or the fusion protein according to any one of claims 4 to 7; or the nucleotide of claim 8.
  10. 10. The composition of claim 9, wherein the thrombotic disorder is Thrombotic Microangiopathy (TMA).
  11. 11. The composition of claim 10, wherein the Thrombotic Microangiopathy (TMA) is selected from the group consisting of thrombocytopenic purpura (TTP), hemolytic Uremic Syndrome (HUS), HELLP (hemolysis, elevated liver enzymes, and reduced platelet count) syndrome, preeclampsia, and sickle cell disease.
  12. 12. A method for screening for ADAMTS13 variants with increased escape rate against autoantibodies comprising the steps of:
    (a) Preparing an ADAMTS13 variant in which one or more amino acid residues selected from the group consisting of: SEQ ID NO:1, at residue 85, at residue 93, at residue 126, at residue 135, at residue 278, at residue 282, at residue 308, at residue 314, at residue 317, at residue 334, at residue 364, at residue 376, at residue 413, at residue 427, at residue 452, at residue 465, at residue 567, at residue 578, at residue 585, at residue 589, at residue 607, at residue 608, at residue 609, at residue 612, at residue 618, at residue 624, at residue 635, at residue 643, at residue 650, at residue 651, at residue 655, at residue 656, at residue 658, at residue 664 and at residue 672; and
    (b) Contacting an autoantibody to ADAMTS13 with the ADAMTS13 variant prepared in step (a) above;
    wherein when the autoantibody binds to the ADAMTS13 variant with less affinity than to wild-type ADAMTS13, the ADAMTS13 variant is determined to be an ADAMTS13 variant with increased escape rate to the autoantibody.
  13. 13. The method of claim 12, wherein step (a) above is performed by replacing one or more amino acid residues selected from the group consisting of: residue 85, residue 93, residue 126, residue 135, residue 278, residue 282, residue 308, residue 314, residue 317, residue 334, residue 376, residue 413, residue 427, residue 465, residue 567, residue 578, residue 585, residue 607, residue 609, residue 612, residue 624, residue 630, residue 643, residue 650, residue 654, residue 656, residue 658 and residue 672 of SEQ ID NO 1.
CN202180077809.2A 2020-11-18 2021-10-25 ADAMTS13 variants with increased escape rate or activity against autoantibodies Pending CN116745426A (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
KR10-2020-0154710 2020-11-18
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PCT/KR2021/014991 WO2022108148A1 (en) 2020-11-18 2021-10-25 Adamts13 variant having increased escaping rate or activity against autoantibody

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ES2382104T3 (en) * 2005-06-17 2012-06-05 Baxter International Inc. Compositions with thrombolytic activity comprising ADAMTS13
EP2374878B1 (en) 2007-06-22 2015-08-19 The Chemo-Sero-Therapeutic Research Institute Novel adamts-13 mutant
CN103429261A (en) * 2010-12-22 2013-12-04 塞法隆澳大利亚股份有限公司 Modified antibody with improved half-life
WO2013096793A1 (en) * 2011-12-21 2013-06-27 The Children's Hospital Of Philadelphia Gain-of-function adamts13 variants resistant to autoantibody inhibition and methods of use thereof
AU2013203062C1 (en) * 2013-03-15 2018-06-28 Takeda Pharmaceutical Company Limited Subcutaneous administration of adamts13
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